The purpose and scope of this project is subdivided in three specific aims that are detailed below: Specific Aim 1. Development of Radiation Induced and Targeted Chemotherapy (RITCH). The concept envisions a non-toxic pro-drug that when administered intravenously will distribute throughout the body. When the pro-drug is subjected to localized electromagnetic radiation it will undergo a chemical transformation into a cytotoxic compound at the site of the tumor. We have used as a prototype RITCH compound the hydrophobic membrane probe Iodonaphthyl-azide (INA), which upon light irradiation undergoes a covalent reaction with transmembrane portions of membrane proteins. We have examined the reactivity of RITCH compounds in vitro and in vivo using various modes of triggering that include light, sono-cavitation and X-ray radiation The animal studies involve pharmacokinetics, bio-distribution and toxicity using the small animal imaging facility at NCI-Frederick. We have identified two new energetic azide derivatives that respond to ultrasound and bind to tissues. We optimized the protocol for delivery and retention of the ultrasound reactive compound eosin-azide to tumors in mice. We discovered that tumor specific hydrogen peroxide can induce binding of eosin-azide to tumor tissue and thus can be used to target azido compound to tumors without the involvement of ultrasound or any other source of energy.Specific Aim 2: Develop Multifunctional Liposomes with Targeting, Imaging and Drug Delivery Capabilities We are developing lipid-based nanoparticles (liposomes) bearing targeting and on-demand drug release properties for improved delivery of cancer therapeutics. For light-triggered applications, we have designed liposomes from a photopolymerizable diacetylenic phospholipid (DC8,9PC), based on its unique partitioning in the liposome membrane. Our formulations also include a tunable aqueous photo-sensitizer to promote electromagnetic radiation-triggered drug release. For biological applications (localized drug delivery), we have used a visible light source (514 nm laser) that does not affect cell viability. Pre-exposure of liposome/cell suspensions to the laser results in improved efficiency of doxorubicin delivery to cells (based on cytotoxicity assays). Biodistribution studies of these liposomes in mice indicate uptake by liver and spleen with no apparent signs of toxicity up to three weeks post injection. Using a nasophareangeal carcinoma model (KB), we show that liposomes accumulate in tumors. Ex-vivo phototriggering in tumor tissue sections by 254 nm (UV) treatment indicate disruption of liposomes and release of contents into the tumor. Specific Aim 3 Elucidate mode of action of membrane-active anti-cancer designed beta-hairpin peptides. We found that the designed model peptides fold at the surface of phosphatidyl serine-containing liposomes causing release of encapsulated molecules of limited size by forming multimeric pores.MD simulations indicate that the peptides share a common subunit organization with Protegrin-1 and Alzheimer amyloid ion channels. The fact that the peptides share the beta-sheet morphology in common with naturally occurring toxic peptides lends credence to the universality of this mechanism. Thus we have applied the discipline of synthetic biology in order to learn more how naturally occurring toxic peptides work.